Fig. 3: Genome-Wide SPLiCR-Seq Screens Identify Regulators of IRE1α-XBP1 Signaling Under Different ER Stress Conditions.
A Schematic of the genome-wide SPLiCR-seq screen workflow. CRISPRi-HEK293T cells were transduced with a genome-wide sgRNA library targeting 11,120 genes that are expressed in HEK293T cells (MOI < 0.3). Following puromycin selection and cell expansion, cells were treated with either Tg or Tm for 24 h to induce ER stress. RNA was extracted and SPLiCR libraries were prepared for NGS. B, C Volcano plots showing gene knockdown phenotypes from genome-wide screens in HEK293T cells treated with Tg (B) or Tm (C). LFC and P values were calculated using MAGeCK75. Key known regulators of the IRE1α-XBP1 pathway are labeled, with PPP1R15A, a novel regulator investigated in this study, highlighted in bold. D Comparison of gene knockdown phenotypes between Tg and Tm screens, showing a strong overlap in common regulators of XBP1 splicing. E, F Functional categorization of hits from the genome-wide screens under Tg (E) and Tm (F) treatment. Red circles, positive hits; blue circles, negative hits. Genes involved in protein homeostasis and ER function were identified in both screens, whereas ER–Golgi trafficking genes were unique to the Tg screen. G GO Biological Process enrichment analysis of the top 100 positive hits (red) and top 100 negative hits (blue) for the Tg (left) and Tm (right) screens. P values were calculated using the Fisher’s Exact test. Source data are provided as a Source Data file.
